The Protective Effect of Thai Fermented Pigmented Rice on Urethane Induced Somatic Mutation and Recombination in Drosophila Melanogaster
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Journal of Medicinal Plants Research Vol. 7(2), pp. 91-98, 10 January, 2013 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.1024 ISSN 1996-0875 ©2013 Academic Journals Full Length Research Paper The protective effect of Thai fermented pigmented rice on urethane induced somatic mutation and recombination in Drosophila melanogaster Yossaporn Plaitho, Kaew Kangsadalampai* and Monruedee Sukprasansap Institute of Nutrition, Mahidol University, Nakhon Pathom 73170, Thailand. Accepted 25 September, 2012 This study was aimed to proof that fermentation could increase phenolic and anthocyanin contents, antioxidant activities and antimutagenicity of Thai pigmented rice. Six cultivars of rice (Sung Yod, Mon Poo, Hom Mali Daeng, Hom Nil, Riceberry, and black glutinous rice) were fermented with a mixed culture of yeasts and molds (Look-Pang) at 30°C for 3 days to produce six types of fermented rice (Khao-Mak). Each sample exhibited significantly higher total phenolic and anthocyanin contents as well as antioxidant activities, including 2,2 ′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), than that of the unfermented one of the same variety. Neither fermented nor unfermented pigmented rice substituted for corn flour in the fly medium was mutagenic in the somatic mutation and recombination test using Drosophila melanogaster . Interestingly, the antimutagenicity of fermented rice against urethane in the same testing was proven to be greater than that of its corresponding unfermented rice. This might be due to the higher content of total phenolic compounds, anthocyanins and antioxidant activities in fermented rice probably because of the catalytic action of enzymes produced by the starter organisms during fermentation which are capable of hydrolyzing glucosides of the inactive components to the active aglycones. Key words: Fermented rice, total phenolic, anthocyanin, antioxidant activities, antimutagenicity. INTRODUCTION Rice is a staple food and an economic crop of Thailand. It Khao-Mak is a traditional fermented rice in Thailand is not only white rice but also many special rice cultivars that is made of white glutinous rice fermented with Look- that contain color pigments, such as black glutinous rice Pang at room temperature for 3 days (Lotong, 1992). (black rice), Hom Nil rice (purple rice) and Sung Yod rice Look-Pang is a microbial starter containing a mixed (red color). Vichapong et al. (2010) revealed that varieties culture of Aspergillus species, Rhizopus species and of Thai pigmented rice such as black glutinous rice and Mucor species, together with Saccharomyces cerevisiae Hom Mali Daeng had higher phenolic compounds, total and Candida species inoculums in rice flour mixed with flavonoid and antioxidant activity than normal white staple herbs such as pepper, garlic and galangal as rice. Anthocyanins are commonly a group of pigments antibacterial agents (Manosroi et al., 2011). Enzymes found in pigmented rice such as purple, black and red from the molds hydrolyze starch in the rice to sugars, rices. These compounds have many biological properties which are partially fermented to alcohol by the yeast. such as scavenging free radicals (Wang and Jiao, 2000), Organic acids (e.g. lactic acid) are also produced prevention of DNA damage in cancer cells (Hou, 2003), (Lotong, 1992). The product gives sweet taste, a little activating glutathione-related enzymes and alcohol and lactic acid flavor, soft texture, with lumps of NAD(P)H:quinone reductase (Shih et al., 2007; Singletary cooked glutinous rice and succulent grain. Black et al., 2007). glutinous rice is sometimes substituted for white glutinous rice to produce Khao-Mak since it is a rich source of phytochemicals such as anthocyanins (Sompong et al., 2011). Interestingly, fermented black glutinous rice *Corresponding author. E-mail: [email protected]. Tel: increased its antimutagenicity against in vivo formed +66 2 800 2380/399. Fax: +66 2 441 9344. nitrosomethylurea in somatic mutation and recombination 92 J. Med. Plants Res. test (SMART) (Vipassanatham et al., 2012) and nitrite compounds) with dimethylsulphoxide (DMSO) (10 ml) for 30 min in treated 1-aminopyrene on Salmonella typhimurium TA98 a shaking water bath (30°C) according to the method of Omata et (Sadabpod et al., 2010) compared with that of its al. (2009). Then, the mixture was centrifuged at 2500 ×g for 10 min and the supernatant was collected. The residue was re-extracted corresponding raw and cooked rice. under the same condition. The combined supernatant was In this investigation urethane or ethyl carbamate was determined for its antioxidant activities using the DPPH assay as used as a positive mutagen in the somatic mutation and described by Fukumoto and Mazza (2000), and the ferric reducing recombination test as suggested by Abraham and Graf antioxidant power (FRAP) assay according to the procedure (1996). It is a natural constituent of tobacco leaves and described by Griffin and Bhagooli (2004). The total phenolic content of the extract was determined according to the method described tobacco smoke (Schmeltz et al., 1978) and is also by Amarowicz et al. (2004). The anthocyanin content of each present in fermented food products such as bread, yogurt sample extracted with acidified ethanol was determined according and cheese (Ough, 1976; Canas et al., 1989; Miller and to the method described by Abdel-Aal and Hucl (1999). Miller, 1983). In rodents, urethane was found to produce lymphomas, lung tumors, hepatomas and melanomas (Mori et al., 2000; Mirvish, 1968; International Agency for SMART Research on Cancer (IARC), 1974) and was also found Two strains of Drosophila melanogaster were used. Males of to induce point mutation, gene conversion, mwh/mwh and females of ORR;flr 3/In(3LR) TM3, ri p p sep l(3)89Aa intrachromosomal recombination, chromosomal bx 34e e Bd S, Ser were obtained from the Institute of Toxicology, aberration and sister chromatid exchanges in yeast, plant Swiss Federal Institute of Technology and University of Zurich. The system and mammalian cells (Schlatter and Lutz, 1990; test was performed as described by Graf et al. (1984). One hundred 3 days old trans-heterozygous larvae ( mwh flr +/mwh TM3 ) were Uggla and Busk, 1992). IARC classified urethane as transferred to the Drosophila medium (Roberts, 1986) in which each possibly carcinogenic to humans (group 2B) (IARC, sample was substituted for corn flour (used for the mutagenicity 1974). Therefore, the objective of this study was to evaluation) or the Drosophila medium in which each sample was determine the total phenolic content, anthocyanin content substituted for corn flour and 20 mM urethane was substituted for and antioxidant activities of fermented pigmented rice as water (used for antimutagenicity evaluation). The standard medium well as the possible antimutagenicity of different types of was used as the negative control while the medium containing 20 mM urethane was used as the positive control. Each experiment fermented pigmented rice in the somatic mutation and was done twice. recombination test. The antimutagenicity of each sample was determined from the percentage of inhibition calculated as follows: percentage of inhibition = ((a-b)/a)x100, where “a” is the frequency of spots MATERIALS AND METHODS induced by urethane alone and b is the frequency of spots induced Chemicals by urethane in the presence of the sample. It is proposed that percentages of inhibitions less than 20, 20-40, 40-60 and more than Urethane, 2, 4, 6-tripyridyl-s-triazine (TPTZ), ferric chloride 60 are the evidences of negligible, weak, moderate and strong hexahydrate and ferrous sulfate heptahydrate were purchased from antimutagenicity, respectively as suggested by Kruawan et al. Sigma Chemical (St. Louis, MO, USA). Diethylether and sodium (2012). acetate trihydrate were purchased from Merck (Darmstadt, Germany). Propionic acid, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid and Folin-Ciocalteu reagent were supplied from Fluka Statistical analysis Chemika (Buchs, Switzerland). Trolox was bought from Aldrich Chemical (Milwaukee, WI, USA). All other chemicals and reagents Total phenolics, anthocyanins and antioxidant activities data were were of analytical grade. reported as means + standard deviations for triplicate analyses of the same sample. One-way analysis of variance (ANOVA) and Schefee’s multiple comparison tests were carried out to test for Preparation of fermented pigmented rice differences among the means of fermented rice using Statistical Package for Social Sciences (SPSS, version 13). The mean values Six cultivars of pigmented unpolished rice, namely, Sung Yod (red of unfermented rice and those of fermented rice were analyzed by rice), Mon Poo (red rice), Hom Mali Daeng (red rice), Hom Nil paired-samples t-test. Statistical significance was considered for p (purple rice), Riceberry (purple rice) and black glutinous rice (black less than 0.05. For SMART, the wings of the surviving flies were rice) were obtained from local markets in Bangkok. A commercial analyzed for the occurrence of mutant spots and evaluated as starter called Look-Pang was bought from ‘100 Year Samchuck described by Frei and Würgler (1988). The estimation of spot Market’ in Suphanburi province. Rice was fermented in Thai frequencies and confidence limits of the estimated mutation traditional style as suggested by Lotong (1992) with slight frequency were performed with significance level of α = β = 0.05. modification as the following. It was mixed with distilled water (1:2 w/v) and cooked in an electric cooker. Cooked rice was cooled at room temperature and fermented with Look-Pang (0.2 g/100 g of raw rice) at 30°C for 3 days in a glass container. The fermented rice RESULTS AND DISCUSSION was dried at 60°C in a hot air oven for 24 h and was stored under vacuum in a polyethylene bag at 4°C before use. Total phenolic and anthocyanin contents The total phenolic content of fermented rice was higher Antioxidant activities, total phenolic and anthocyanin contents than that of its corresponding unfermented one (Figure of fermented pigmented rice 1A).